Oncological diseases are the second most common cause of death after cardiovascular diseases [http://www.who.int/healthinfo/EN_WHS2012_Full.pdf]. According to different sources, in the last 10 years the number of people diagnosed with cancer increased by 15%. In Russian Federation, more than 490 thousand new patients are registered annually, 2.5 million cancer carriers are under the care of a doctor, and more than 290 thousand of cancer-stricken die from different cancer forms every year. For this reason, the development and creation of safe, effective and affordable antitumor pharmaceuticals is essential.
One type of antitumor pharmaceuticals refers to products of biotechnological synthesis—monoclonal antibodies (MAbs). Several pharmaceuticals are currently in use, among them there are MAbs to epidermal growth factor receptor, to vascular endothelial growth factor, which are proven to have an effect on tumor cells activity and possess an insufficient influence on the functioning of normal cells of different tissues [Zhukov N. V., Tjulandin S. A. Targeted therapy in the treatment of solid tumors: practice contradicts theory/Biochemistry (Mosc). 2008 May; 73(5):605-18].
In the world approximately 10 antitumor pharmaceuticals based on monoclonal antibodies are registered and produced currently, notably rituximab (MAb to CD20 receptor), trastuzumab (HER2neu receptor is the target), bevacizumab (VEGF), cetuximab and panitumumab (EGFR). At present, no registered pharmaceuticals exist which target a surface antigen syndecan-1 (CD138).
Syndecan-1 protein (CD138) [Wijdenes J., Dore J. M., Clement C., Vermot-Desroches C. (2003). “CD138”. J. Biol. Regul. Homeost. Agents 16 (2): 152-5] is a promising target for antitumor therapy with monoclonal antibodies: it is proven that this molecule plays an important role in the tumor cells activity and it is revealed that it possesses an insufficient influence on the functioning of the normal cells of various tissues [Guo P. et al. Expression of legumain correlates with prognosis and metastasis in gastric carcinoma//PloS One. 2013. Vol. 8, No 9. P. e73090].
This molecule provides cell-cell and cell-matrix interactions, adhesion and migration. Overexpression of CD138 was revealed in the majority (approximately 90%) of cells of various tumor types, including breast, colon, gastric, prostate cancer and of other epithelial malignancies [Rousseau et al. EJNMMI Research 2011, Bayer-Garner I. B., Sanderson R. D., Dhodapkar M. V. et al. Syndecan-1 (CD138) immunoreactivity in bone marrow biopsies of multiple myeloma: shed syndecan-1 accumulates in fibrotic regions. Mod Pathol 2001; 14:1052-8]. Such phenomenon as a loss of syndecan-1 from the cell surface of myeloid cells and its accumulation in stroma, as a rule, in fibrotic regions, is also described [Baikov V. V. Difficulties in morphological diagnostics of multiple myeloma. Moscow, Oncohematology 2'2007, p.p. 10-15]. It is revealed that under the loss of syndecan-1 myeloid cells undergo TRAIL-induced apoptosis [Wu Y. H., Yang C. Y., Chien W. L., Lin K. I., Lai M. Z. Removal of syndecan-1 promotes TRAIL-induced apoptosis in myeloma cells. J Immunol. 2012 Mar 15; 188(6):2914-21. Epub 2012 Feb 3]. Syndecan-1 is connected functionally with the invasive growth and metastatic propensity of the tumor cells. In terms of the malignancies therapy the activity of CD138 is associated with such properties of tumor cells, which almost do not respond to existing therapies.
The localization of CD138 on the surface of the tumor cells in the form, which can be recognized by antibodies, is revealed [Wu B. et al. Blastocystis legumain is localized on the cell surface, and specific inhibition of its activity implicates a pro-survival role for the enzyme//J. Biol. Chem. 2010. Vol. 285, No 3. P. 1790-1798]. Moreover, the data exist, which reveal the effectiveness of vaccination using CD138 as an antigen [Bae J., Tai Y. T., Anderson K. C., Munshi N. C. 2011].
The murine antibodies to syndecan-1 are currently in use—B-B4 (IgG1) [Wijdenes J., Vooijs W. C., Clement C. et al. A plasmacyte selective monoclonal antibody (B-B4) recognizes syndecan-1. Br. J. Haematol 1996; 94:318-23], B-B2 (IgG2b), as well as chimeric antibodies [Baikov V. V. 2007, Kovrigina A. M., Probatova N. A. Differential diagnostics of non-Hodgkin's B-cell's lymphoma. Oncohematology. 2′2007, p.6]—MI15 (IgG1κ)—and their conjugates with tags [ClinicalTrials.gov identifier: NCT01296204, Gattei V., Godeas C., Degan M., Rossi F. M., Aldinucci D., Pinto A. Characterization of anti-CD138 monoclonal antibodies as tools for investigating the molecular polymorphism of syndecan-1 in human lymphoma cells. Br J Haematol. 1999 January; 104(1):152-62]. Such compounds are used for the purpose of research or diagnostics, the antibody performing only the targeting function [Baikov V. V. 2007, Kovrigina A. M., Probatova N. A., 2007]. The cytotoxic functions are performed by the effector molecules, which are mostly conjugated with the antibody [WO2009080832 (A1), WO2009080830 (A1), WO2010128087 (A2)]. Murine and rabbit MAbs are recognized by human immune system as foreign because of differences in the constant domains of the antibodies (CH, CL), and an immune response against them is elicited. For the purpose of use in humans, chimeric antibodies are being developed, which possess human constant domains (CH, CL) and murine variable domains (VH, VL). This modification allows making the MAbs safer. Chimeric monoclonal antibody to CD138 of subclass IgG4 is known [WO2009080829 (A1)], which is considered the prototype by the authors of the present invention. However, this MAb provides only targeting of a cytotoxic agent conjugated thereto (DM4).
A number of inventions is known, where antibodies to CD138 are mentioned, but the structure of these antibodies is not given. Antibody to human CD138 is known having a modification of glycosylation, obtained with the use of a cell expressing at least one nucleic acid encoding (1,4)-N-acetylglucosaminiltransferase III (GnT III), and at least one transfected nucleic acid encoding human anti-CD138 antibody, but the amino acid and/or nucleotide sequences of such antibody are not given, the structure of such antibody is not characterized [RU2321630C2]. A variant of invention is known, wherein the anti-CD138 antibody is co-administered with a conjugate of anti-Trop-2 antibody and a chemotherapeutic agent or a conjugate of antibody to mucin or its antigen-binding fragment and a radionuclide for the treatment of pancreatic cancer [US2014044640 (A1)]. Conjugate is known of antibody, which binds to CD138, and of anti-CD74 antibody [US2014056917A1], and of SN38 molecule [US2014058067 (A1)], including fragments thereof, as part of an antibody bispecific structure [US2014086832 (A1)]. Composition is also known containing an anchor region capable of binding to a plasma cell and a site binding to specific plasma cell antibodies which is bound to the anchor region, an antibody capable of binding to syndecan-1 may be a part of the design [WO2014037519 (A2)].
A recombinant antibody to syndecan-1 is currently in use (OC-46F2), produced as a result of in vitro phage-display library selection (ETH-2-Gold) of human antibodies on human melanoma cells and subsequent expression of the obtained genes in mammalian cells [Orecchia P, Conte R, Balza E, Petretto A, Mauri P, Mingari M C, Carnemolla B. A novel human anti-syndecan-1 antibody inhibits vascular maturation and tumour growth in melanoma. Eur J Cancer. 2013 May; 49(8):2022-33. doi: 10.1016/j.ejca.2012.12.019. Epub 2013 Jan. 24]. It has been revealed that this antibody inhibits vessels maturation and tumor growth in the experimental human melanoma model (on mice). It has also been proven to be therapeutically effective in the human ovarian carcinoma model.
OC-46F2 represents a single-chain variable fragment (scFv)—a fusion protein consisting of variable fragments of a heavy (VH) and a light (VL-λ) immunoglobulin chains joined via a short linker peptide.
This compound does not contain constant antibody fragments, which complicates the adequate immune response associated with the use of antibodies formation, —the variable fragments of the molecule provide only junction with the antigen, determining the deterrent effect revealed by the authors. However, additional active compounds and, possibly, a production of conjugates are necessary for the tumor elimination in this case.
In such a way, the currently described murine antibodies to CD138 are not suitable for use in humans because of their high immunogenicity. They are used for laboratory purposes only. Chimeric MAbs do not possess any independent therapeutic effect—they are applied only as a targeting agent—and can also be immunogenic. An individual reaction of a patient to a particular MAb should not be ruled out. Besides, the active agent, targeted via antibody, or products of its degradation can become a stress factor for the human organism due to their structure, ability to decay, the decay period, and a character of compounds, to which it degrades. The exact structure of the natural human monoclonal antibodies to syndecan-1 is not established.
Taking into consideration the functional role of syndecan-1 in the invasive growth and metastatic propensity of the tumors, generation of more efficient and safe antibodies to it is a critical task at present.
This task is solved by the present invention which does not possess disadvantages of analogs.
The technical result of application of the present antibody specific to CD138 is at least in a spectrum spreading of MAbs, also to CD138, used in a tumor therapy, which allows providing treatment under idiocrasy or weak tolerance to analogs.
The technical result is in an increase of efficacy of the antibody to CD138. The revealed technical result is achieved by obtaining independent antitumor activity due to the use of the calculated and created by the authors proposed antibody of IgG4 isotype, represented by a particular combination of described sequences of antibody chains obtained by the inventors, as well as by the increased affinity to the antigen, also due to the use of the present antibody, also due to humanization of the antibody: due to the use of human antibody frameworks, it is possible to position the hypervariable regions more precise, which increases the correspondence of epitope and paratope, and therefore affinity.
The independent antitumor activity is achieved in one of embodiments of the invention by the fact that fragments of IgG3 chains mediating high antibody- and complement-dependent cytotoxicity are inserted into the constant fragment of the heavy chain of the IgG4 antibody, as a result such an antibody has an increased cytotoxic effect upon binding to CD138. Said technical result when using this embodiment of the invention is also achieved by the increase in antibody half-life period, which is achieved by introducing amino acid substitutions at the C-terminus of the constant part of the heavy chain. This allows achieving a longer effect, to use a lower dosage, perform fewer administrations of the antibody, which may also allow to reduce the cost of treatment.
The technical result of application of the present antibody to CD138 is also in an increase of safety of the anticancer drug. The indicated technical result is achieved by reducing immunogenicity due to the humanization of the antibody and also due to the fact that the active agent is a monoclonal antibody molecule—a protein, which degrades to amino acids afterwards, h.e. if applied, any consequences of nonclearance of an active agent (a nonprotein chemical compound) or products of its degradation within a certain time are excluded, as a result the decrease of stressing effect of the antitumor pharmaceutical on human organism is observed.
The technical result is also in an increase of efficacy, in simplification and fastening of the production, which allows lowering the price, which is achieved by obtaining higher expression rate of the antibody in mammalian cells at a short time due to codon optimization of the coding sequences, as well as to the insertion of signal secretion sequence at the N-terminus of the light and heavy chains.